Multifunctional amine capture agents

ABSTRACT

Multifunctional compounds having acylsulfonamide amine-reactive groups are described that can be used for the capture of amine containing materials.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 11/015,543 filed onDec. 17, 2004.

BACKGROUND

Amine-containing materials, such as amine-containing analytes, aminoacids, DNA fragments, RNA fragments, protein fragments, organelles, andimmunoglobins, immobilized on the surface of a substrate can be used innumerous applications. The covalent attachment of amine-containingmaterials to a substrate can be accomplished, for example, by thereaction of the amine with a reactive group on the surface of thesubstrate. This amine-reactive functional group can be, for example, anactivated acyl derivative, such as an N-hydroxy succinimide ester; or,an active cyclic acyl compound, such as azlactone. A stable amide bondis formed from reaction of the amine with the active acyl group, eitherwith expulsion of N-hydroxy succinimide or opening of the azlactone.

Although a wide variety of amine-reactive compounds of the typedescribed above, with an amine capturing functional group on one end anda different substrate anchoring functional group on the other end of adivalent linking group, can be conveniently synthesized, many suchmodifications require difficult separations to achieve the necessaryselectively substituted compound. For example, longer or oligomericdivalent linking groups, or, especially, branched or multi-functionallinking groups, would be difficult to prepare having a single, substratespecific, reactive functional group on only one end of the molecule. Analternative approach involves converting some or all functional groupsto amine capture functional groups. Such molecules could be beneficialfor efficient attachment, as well as for control of the activity of theimmobilized amine-containing material, especially in biological systems.

Additionally, some surfaces may have few or no complementary functionalgroups for anchoring amine compounds. Such inert surfaces are oftenconveniently functionalized by treatment with a surface aminating agent,such as amino alkyl silanes or polyethylene imine. Subsequent exposureof these aminated surfaces to solutions of excess di- ormulti-functional amine-capture compounds would convert the surface ofthe substrate into a surface with grafts of amine-reactive functionalgroups.

Thus, there exists a need for compounds for reaction withamine-functional surfaces having di- and multi-functionalamine-reactive, terminal functional groups.

SUMMARY

The present invention provides multi-functional compounds with terminalacyl sulfonamide groups. These compounds can be used to treatamine-functional surfaces. Such compounds can also be used ascrosslinkers for amine containing materials to provide gels andhydrogels, for example.

The compounds are of the formula:

(A-)_(y)-Q

wherein A is independently selected from the group consisting offunctional groups having the following formulas:

wherein R¹, R², R³, R⁴, R⁵, R⁶, Y¹, Y², Y³, y, Q, and Z are definedherein below, with the proviso that Q, Y¹, Y², and Y³ are free ofdisulfide groups.

DEFINITIONS

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

As used herein, the term “alkyl” refers to a monovalent radical of analkane and includes groups that are linear, branched, cyclic, orcombinations thereof. The alkyl group typically has 1 to 30 carbonatoms. In some embodiments, the alkyl group contains 1 to 20 carbonatoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbonatoms. Examples of alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, cyclohexyl, n-heptyl, n-octyl, and ethylhexyl.

As used herein, the term “alkylene” refers to a divalent radical of analkane. The alkylene can be straight-chained, branched, cyclic, orcombinations thereof. The alkylene typically has 1 to 200 carbon atoms.In some embodiments, the alkylene contains 1 to 100, 1 to 80, 1 to 50, 1to 30, 1 to 20, 1 to 10, 1 to 6, or 1 to 4 carbon atoms. The radicalcenters of the alkylene can be on the same carbon atom (i.e., analkylidene) or on different carbon atoms.

As used herein, the term “aralkyl” refers to a monovalent radical of thecompound Ar—R where Ar is an aromatic carbocyclic group and R is analkyl group.

As used herein, the term “aralkylene” refers to a divalent radical offormula —R—Ar— where Ar is an arylene group and R is an alkylene group.

As used herein, the term “aryl” refers to a monovalent aromaticcarbocyclic radical. The aryl can have one aromatic ring or can includeup to 5 carbocyclic ring structures that are connected to or fused tothe aromatic ring. The other ring structures can be aromatic,non-aromatic, or combinations thereof. Examples of aryl groups include,but are not limited to, phenyl, biphenyl, terphenyl, anthryl, naphthyl,acenaphthyl, anthraquinonyl, phenanthryl, anthracenyl, pyrenyl,perylenyl, and fluorenyl.

As used herein, the term “arylene” refers to a divalent radical of acarbocyclic aromatic compound having one to five rings that areconnected, fused, or combinations thereof. In some embodiments, thearylene group has up to 5 rings, up to 4 rings, up to 3 rings, up to 2rings, or one aromatic ring. For example, the arylene group can bephenylene.

The above aryl and arylenes can optionally contain substituents such aslower alkyl, halo, and alkoxy.

As used herein, the term “carbonyl” refers to a divalent group offormula —(CO)—.

As used herein, the term “carbonylimino” refers to a divalent group offormula —(CO)NR^(a)— where R^(a) is hydrogen, alkyl, or aryl.

As used herein, the term “carbonyloxy” refers to a divalent group offormula —(CO)O—.

As used herein, the term “chloroalkyl” refers to an alkyl having atleast one hydrogen atom replaced with a chlorine atom.

As used herein, the term “disulfide” refers to a divalent group offormula —S—S—.

As used herein, the term “fluoroalkyl” refers to an alkyl having atleast one hydrogen atom replaced with a fluorine atom. Some fluoroalkylgroups are perfluoroalkyl groups.

As used herein, the term “heteroalkylene” refers to a divalent alkylenehaving one or more carbon atoms replaced with a sulfur, oxygen, orNR^(d) where R^(d) is hydrogen or alkyl. The heteroalkylene can belinear, branched, cyclic, or combinations thereof and can include up to400 carbon atoms and up to 30 heteroatoms. In some embodiments, theheteroalkylene includes up to 300 carbon atoms, up to 200 carbon atoms,up to 100 carbon atoms, up to 50 carbon atoms, up to 30 carbon atoms, upto 20 carbon atoms, or up to 10 carbon atoms.

As used herein, the term “heteroarylene” refers to a divalent arylenehaving one or more carbon atoms replaced with a sulfur, oxygen, orNR^(f) where R^(f) is hydrogen or alkyl.

As used herein, the term “oxy” refers to a divalent group of formula—O—.

As used herein, the term “perfluoroalkyl” refers to an alkyl group inwhich all of the hydrogen atoms are replaced with fluorine atoms.

As used herein, the term “thio” refers to a group of formula —S—.

As used herein, the term “room temperature” refers to a temperature ofabout 20° C. to about 25° C. or about 22° C. to about 25° C.

As used herein, a curve connecting two groups in a formula indicatesthat the two groups together form part of a cyclic structure.

For any of the compounds presented herein, each one of the followingvariables (e.g., R¹, R², Y¹, Y², Z, A, and so on) in any of itsembodiments can be combined with any one or more of the other variablesin any of their embodiments as would be understood by one of skill inthe art. Each of the resulting combinations of variables is anembodiment of the present invention.

When a group (or substituent or variable) is present more than once in acompound or polymer described herein, each group (or substituent orvariable) is independently selected, whether explicitly stated or not.For example, for the formula (A)_(y)-Q each A group is independentlyselected. Furthermore, when more than one A group is present and each Agroup contains one or more L groups, as defined below, then each L groupis also independently selected.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides compounds of the formula:

(A-)_(y)-Q

wherein each A is independently selected from the group consisting offunctional groups having the following formulas:

wherein R¹, R², R³, R⁴, R⁵, R⁶, Y¹, Y², Y³, y, Q, and Z are definedherein below, with the proviso that Q, Y¹, Y², and Y³ are free ofdisulfide groups. Such A groups are preferably terminal groups.

Herein, in the compounds of the formula (A-)_(y)-Q, Q is a single bondor a y-valent atom or group. In certain embodiments, Q is an atomselected from C, N, S, O, or P. In certain embodiments, Q is a y-valentgroup containing up to 20 carbon atoms and up to 6 heteroatoms and/orfunctional groups (such as carbonyl groups). In certain embodiments, Qincludes a ring system. Exemplary Q groups include carbonyl, alkylenes,alkanetriyl (i.e., a trivalent radical of an alkane), heteroalkylenes,arylenes, heteroarylenes, alkylene-oxy-alkylenes (e.g., —CHCH₂OCH₂CH—),alkylene-carbonyl-alkylenes, and combinations thereof (e.g., groupsincluding both alkylene and arylene groups with or without heteroatomsand/or functional groups). Exemplary Q ring structures include thefollowing:

Herein, y is an integer of 2 to 10. In certain embodiments, y is aninteger of 2 to 6. In some embodiments, y is an integer of 2 to 4. Insome embodiments, y is an integer of 2 to 3. In some embodiments, y is 2and the A groups are terminal.

The A groups may be the same or different. For synthetic convenience,however, they are often the same.

Herein, in Formula I, R¹ and R² together with a dicarboximide group towhich they are attached form a four to eight membered heterocyclic orheterobicyclic group that can be fused to an optional aromatic group,optional saturated or unsaturated cyclic group, or optional saturated orunsaturated bicyclic group.

Herein, in Formula II, R³ is an alkyl, aryl, aralkyl, or —NR^(a)R^(b)wherein R^(a) and R^(b) are each an alkyl group or taken together withthe nitrogen atom to which they are attached form a four to eightmembered heterocyclic group.

In certain embodiments of Formula II, R³ is an alkyl, aryl, or aralkylgroup. Suitable alkyl groups typically contain no greater than 30 carbonatoms, no greater than 20 carbon atoms, no greater than 10 carbon atoms,no greater than 6 carbon atoms, or no greater than 4 carbon atoms. Insome compounds, the alkyl group is methyl, ethyl, or propyl. Suitablearyl groups typically contain 6 to 18 carbon atoms, 6 to 12 carbonatoms, or 6 carbon atoms. In some compounds, the aryl group is phenyl.An example of an aryl group is 4-methylphenyl. Suitable aralkyl groupstypically contain an aryl group having 6 to 30 carbon atoms and an alkylgroup having no greater than 30 carbon atoms.

In other embodiments of Formula II, R³ is a group —NR^(a)R^(b) whereR^(a) and R^(b) are alkyl groups having no greater than 10 carbon atoms,no greater than 6 carbon atoms, or no greater than 4 carbon atoms.Alternatively, the R^(a) and R^(b) groups can combine together with thenitrogen atom to which they are attached to form a 4 to 8 membered ringstructure. For example, R^(a) and R^(b) can combine to form a five orsix membered heterocyclic group having a nitrogen heteroatom.

Herein, in Formula II, R⁴ and R⁵ together with a dicarboximide group towhich they are attached form a four to eight membered heterocyclic orheterobicyclic group that can be fused to an optional aromatic group,optional saturated or unsaturated cyclic group, or optional saturated orunsaturated bicyclic group.

Herein, in Formula III, R⁶ is an alkyl, fluoroalkyl, chloroalkyl, aryl,—NR^(c)R^(d) wherein R^(c) and R^(d) are each an alkyl group or takentogether with the nitrogen atom to which they are attached form a fourto eight membered cyclic group, or R⁶ taken together with R^(e) and thegroups to which they are attached form the four to eight memberedheterocyclic or heterobicyclic group that can be fused to the optionalaromatic group, optional saturated or unsaturated cyclic group, oroptional saturated or unsaturated bicyclic group.

In some embodiments of Formula III, R⁶ can be a C₁₋₃₀ alkyl, a C₁₋₁₀alkyl, or a C₁₋₆ alkyl. In other embodiments of Formula III, R⁶ can be aC₁₋₃₀ fluoroalkyl, a C₁₋₁₀ fluoroalkyl, or a C₁₋₄ perfluoroalkyl group.In still other embodiments of Formula III, R⁶ can be a C₆₋₁₂ aryl. Forexample R⁶ can be a phenyl group.

Herein, Z is an alkyl, aryl, or —(CO)R^(e). In some embodiments ofFormula III, Z can be alkyl or aryl. For example, Z can be a C₁₋₆ alkyl.In other examples, Z can be a C₆₋₁₂ aryl. In other embodiments ofFormula I, Z can be a —(CO)R^(e) group, wherein R^(e) together with R⁶and groups to which they are attached form a four to eight memberedheterocyclic or heterobicyclic group having a nitrogen heteroatom and asulfur heteroatom, wherein the heterocyclic or heterobicyclic group canbe fused to an optional aromatic group, optional saturated orunsaturated cyclic group, or optional saturated or unsaturated bicyclicgroup.

Herein, Y¹, Y², and Y³ are each independently a single bond or adivalent group selected from the group consisting of an alkylene,heteroalkylene, arylene, heteroarylene, carbonyl, carbonyloxy,carbonylimino, oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, andcombinations thereof. In certain embodiments, Y¹, Y², and Y³ are eachindependently selected from the group consisting of groups having thefollowing formulas —Y^(1a)—Ar¹— and —Ar¹—Y^(1a)—, wherein: Ar¹ is anarylene; and Y^(1a) is selected from the group consisting of a singlebond, alkylene, heteroalkylene, carbonyl, carbonyloxy, carbonylimino,oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof.

In certain embodiments, Y¹, Y², and Y³ are each independently selectedfrom the group consisting of groups having the following formulas:—Y^(1a)—Ar¹— and —Ar¹—Y^(1a)—. In such formulas, Ar¹ is an arylene(preferably, a phenylene), and Y^(1a) is selected from the groupconsisting of a single bond, alkylene, heteroalkylene, carbonyl,carbonyloxy, carbonylimino, oxy, thio, —NR^(f)— where R^(f) is hydrogenor alkyl, and combinations thereof.

In certain embodiments, Y¹, Y², and Y³ each independently includes afirst alkylene group linked to an arylene group with a group selectedfrom the group consisting of a carbonyl, carbonyloxy, carbonylimino,oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof. In certain of these embodiments, the first alkylene group isfurther linked to a second alkylene or a first heteroalkylene group witha group selected from the group consisting of a carbonyl, carbonyloxy,carbonylimino, oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, andcombinations thereof. In certain of these embodiments, additionalalkylene or heteroalkylene groups can be linked to the second alkyleneor to the first heteroalkylene group with a group selected from thegroup consisting of a carbonyl, carbonyloxy, carbonylimino, oxy, thio,—NR^(f)— where R^(f) is hydrogen or alkyl, and combinations thereof.

In certain embodiments, Y¹, Y², and Y³ each independently includes afirst heteroalkylene group linked to an arylene with a group selectedfrom the group consisting of a carbonyl, carbonyloxy, carbonylimino,oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof. In certain of these embodiments, the first heteroalkylene groupis further linked to a second heteroalkylene or to a first alkylenegroup with a group selected from the group consisting of a carbonyl,carbonyloxy, carbonylimino oxy, thio, —NR^(f)— where R^(f) is hydrogenor alkyl, and combinations thereof. In certain of these embodiments,additional alkylene or heteroalkylene groups linked to the secondheteroalkylene or to the first alkylene group with groups selected fromthe group consisting of carbonyl, carbonyloxy, carbonylimino group, oxy,thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof.

In certain embodiments, Y¹, Y², and Y³ each independently includes afirst alkylene group connected to a second alkylene group or to a firstheteroalkylene group with a group selected from the group consisting ofa carbonyl, carbonylimino, carbonyloxy, oxy, thio, —NR^(f)— where R^(f)is hydrogen or alkyl, and combinations thereof. In certain of theseembodiments, additional alkylene or heteroalkylene groups connected tothe second alkylene group or the first heteroalkylene group with a groupselected from the group consisting of a carbonyl, carbonylimino,carbonyloxy, oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, andcombinations thereof.

In certain embodiments, Y¹, Y², and Y³ each independently includes afirst heteroalkylene group connected to a second heteroalkylene group orto a first alkylene group with a group selected from the groupconsisting of a carbonyl, carbonylimino, carbonyloxy, oxy, thio,—NR^(f)— where R^(f) is hydrogen or alkyl, and combinations thereof. Incertain of these embodiments, additional alkylene or heteroalkylenegroups connected to the second heteroalkylene group or the firstalkylene group.

In certain embodiments, Y¹, Y², and Y³ are each independently aheteroalkylene having, for example, 1-30 carbon atoms and up to 30heteroatoms selected from the group consisting of N, O, S, andcombinations thereof, wherein the heteroalkylene group is linear,branched, cyclic, or combinations thereof.

In certain embodiments, Y¹, Y², and Y³ are each independently analkylene having 1-30 carbon atoms, wherein the alkylene group is linear,branched, cyclic, or combinations thereof. In certain of theseembodiments, the alkylene can be straight chain or branched with 1-20carbon atoms. In certain of these embodiments, the alkylene is of theformula (CH₂)_(n), where n is an integer of 1 to 20.

In certain embodiments, Y¹, Y², and Y³ each independently includes anarylene group (preferably, including up to 18 carbon atoms, up to 12carbon atoms, or up to 6 carbon atoms), in addition to one or morealkylene groups and one or more heteroalkylene groups.

Exemplary Compounds

Exemplary Formula I structures include, but are not limited to, thefollowing:

wherein: R is an alkyl; and Y¹ is the same as previously defined forFormula I. In certain of these exemplary embodiments, Y¹ can be —Y¹—Ar¹—or —Ar¹—Y^(1a)—, wherein Ar¹ is an arylene (preferably, a phenylene),and Y^(1a) is selected from the group consisting of a single bond,alkylene, heteroalkylene, carbonyl, carbonyloxy, carbonylimino, oxy,thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof. The functional groups of Formula I can be unsubstituted orsubstituted with a halo, alkyl, alkoxy, or combinations thereof.

Exemplary Formula I structures also include, but are not limited to, thefollowing:

wherein: R¹ and R² are is the same as previously defined for Formula I;each n is independently an integer of 1 to 100; m is an integer of 1 to200; k is an integer of 2 to 4; D is oxygen, sulfur, or NH; Ar¹ is anarylene group; each L is independently oxygen or NR^(f) where R^(f) ishydrogen or alkyl; and q is an integer of 0 or 1. In such embodiments,preferably, n is no greater than 80, no greater than 60, no greater than40, no greater than 20, or no greater than 10; preferably, m is nogreater than 150, no greater than 100, no greater than 80, no greaterthan 60, no greater than 40, no greater than 20, or no greater than 10;preferably, k is equal to 2; preferably, D is oxygen; and preferably,Ar¹ is phenylene.

Exemplary Formula II structures include, but are not limited to, thefollowing:

wherein R³ and Y² are the same as previously defined for Formula II. Thefunctional groups of Formula II can be unsubstituted or substituted witha halo, alkyl, alkoxy, or combinations thereof.

Exemplary Formula II structures also include, but are not limited to,the following:

wherein: R³, R⁴, and R⁵ are is the same as previously defined forFormula II; v is an integer of 1 to 200; x is an integer of 1 to 4; andD is oxygen, sulfur, or NH. In such embodiments, preferably, v is nogreater than 150, no greater than 100, no greater than 80, no greaterthan 60, no greater than 40, no greater than 20, no greater than 10, nogreater than 5, no greater than 4, no greater than 3, no greater than 2,or equal to 1, and more preferably, v is 1 or 2; preferably, x is nogreater than 3, no greater than 2, or equal to 1, and more preferably, xis 1 or 2; and preferably, D is oxygen or sulfur.

An exemplary Formula III structure includes a heterocyclic group fusedto an aromatic group as shown in the following formula:

wherein Y³ is the same as previously defined for Formula III.

In certain embodiments, the multifunctional compounds of the presentinvention include two or more pendant groups independently selected fromthe following formulas:

wherein: W is C_(k)H_(2k)D or DC_(k)H_(2k); D is oxygen, sulfur, or NH(preferably, oxygen); n is an integer of 1 to 100 (preferably no greaterthan 80, no greater than 60, no greater than 40, no greater than 20, nogreater than 10); m is an integer of 1 to 200 (preferably no greaterthan 150, no greater than 100, no greater than 80, no greater than 60,no greater than 40, no greater than 20, no greater than 10); p is aninteger of 1 to 10 (preferably no greater than 8, no greater than 6, nogreater than 4, or no greater than 2); q is an integer of 0 or 1; t isan integer of 0 to 12 (preferably no greater than 10, no greater than 8,no greater than 6, no greater than 4, no greater than 2, or equal to 0);k is an integer of 2 to 4 (preferably no greater than 3, no greater than2, or equal to 2); and each L is independently oxygen or NR^(f) whereR^(f) is hydrogen or alkyl; with the proviso that at least one L ispresent in each -L_(q)-C(O)-L_(q)- moiety and there are noheteroatom-heteroatom bonds.

In certain embodiments, the multifunctional compounds of the presentinvention include two or more pendant groups independently selected fromthe following formulas:

wherein: n is an integer of 1 to 100 (preferably no greater than 80, nogreater than 60, no greater than 40, no greater than 20, no greater than10); m is an integer of 1 to 200 (preferably no greater than 150, nogreater than 100, no greater than 80, no greater than 60, no greaterthan 40, no greater than 20, no greater than 10); p is an integer of 1to 10 (preferably no greater than 8, no greater than 6, no greater than4, or no greater than 2); t is an integer of 0 to 12 (preferably nogreater than 10, no greater than 8, no greater than 6, no greater than4, no greater than 2, or equal to 0); k is an integer of 2 to 4(preferably no greater than 3, no greater than 2, or equal to 2); each Lis independently oxygen or NR^(f) where R^(f) is hydrogen or alkyl; andq is an integer of 0 or 1.

Preferred multifunctional compounds are difunctional or trifunctionalcompounds of the following formulas:

wherein m′ is an integer of 1 to 200 (preferably no greater than 150, nogreater than 100, no greater than 80, no greater than 60, no greaterthan 40, no greater than 20, no greater than 10).

Methods of Preparation

The functionally substituted amine capture agents of Applicants'Copending patent application Ser. Nos. 10/714,053 and 10/713,174 filedon 14 Nov. 2003, and Ser. Nos. 10/987,075 and 10/987,522 filed on 12Nov. 2004, can be used to make the multifunctional compounds of thepresent invention. This can be done by attaching such compounds to acore Q group bearing y complementary functional groups to give themultifunctional amine capture agents of the present invention. Forexample, ClC(O)C₆H₄SO₂N(C(O)CH₂)₂ (Preparative Example 2) can be reactedwith a diol such as polyethylene glycol, or a triol such astrimethylolpropane ethoxylate. Also, a silane such as(EtO)₃SiC₁₀H₂₂C(O)-saccharin can be pre-reacted with tetraethoxysilaneto form a sol-gel condensate including multiple amine capture acylsaccharin groups. Alternatively, the amine capture group can be formedat the terminus of a multifunctional Q group by the reactionsillustrated in the Applicants' Copending patent application Ser. Nos.10/714,053 and 10/713,174 filed on 14 Nov. 2003, and Ser. Nos.10/987,075 and 10/987,522 filed on 12 Nov. 2004. For example, a Qgroup-containing multifunctional acid chloride can be reacted withsodium saccharin, or a Q group-containing multisulfonamide can bereacted with succinoyl chloride.

Uses

The multifunctional compounds of the invention can be used, for example,for immobilizing amine-containing material (e.g., the multifunctionalcompounds can be attached to a substrate with one functional group andat least one remaining functional group can react with anamine-containing material). In some embodiments, the amine-containingmaterial is an amine-containing analyte. In other embodiments, theamine-containing materials are biomolecules such as, for example, aminoacids, peptides, DNA, RNA, protein, enzymes, organelles, immunoglobins,or fragments thereof. The immobilized amine-containing materials canalso be used for biological separations or for detection of the presenceof various biomolecules. Additionally, the immobilized amine-containingmaterials can be used in bioreactors or as biocatalysts to prepare othermaterials. The substrate-attached multifunctional compound can be usedto detect amine-containing analytes.

Biological amine-containing materials often can remain active afterattachment to the substrate-attached multifunctional compound. Forexample, an immobilized antibody can bind with antigen or an immobilizedantigen can bind to an antibody. An amine-containing material can bindto a bacterium. In a more specific example, the immobilizedamine-containing material can bind to a Staphylococcus aureus bacterium(e.g., the immobilized amine-containing material can be a biomoleculethat has a portion that can specifically bind to the bacterium).

The multifunctional compounds of the present invention can be attachedto a wide variety of substrates, including metals, glasses, polymers,ceramics, etc. The substrates for this use typically have an aminegroup, which can be formed in wet chemistry techniques such as reactinga polyacrylate with an alkylenediamine under forcing conditions or aglass surface with an aminoalkylsilylating agent or by vapor techniquessuch as an ammonia plasma.

The compounds of the invention are particularly useful as crosslinkersto cause precipitation or gellation of amine-containing polymer. Suchsystems can be adhesives and are useful to bond or seal tissue in vivo,as described for naturally occurring polyamines in U.S. Pat. No.5,583,114 and for synthetic polyamines in U.S. Pat. No. 5,874,500. Thesepreviously known systems utilized diesters of N-hydroxysuccinimide asthe amine capture groups. The multifunctional compounds of the presentinvention typically have greater hydrolytic stability than acylN-hydroxysuccinimides and consequently are more useful as gelling andtethering agents in aqueous systems.

EXAMPLES

These examples are merely for illustrative purposes only and are notmeant to be limiting on the scope of the appended claims. All parts,percentages, ratios, etc. in the examples and the rest of thespecification are by weight, unless noted otherwise. Solvents and otherreagents used were obtained from Sigma-Aldrich Chemical Company;Milwaukee, Wis. unless otherwise noted.

Table of Abbreviations Abbreviation or Trade Designation DescriptionEtOAc Ethyl acetate ACN Acetonitrile IPA isopropyl alcohol DMFDimethylformamide PEG 3400 Poly(ethylene glycol) M_(n) about 3400 PEG1000 Poly(ethylene glycol) M_(n) approximately 1000; PEG 600Poly(ethylene glycol) bis(carboxymethyl) ether) diacidHO₂CCH₂(OC₂H₄)_(n)OCH₂COOH M_(n) approximately 600 commerciallyavailable from Fluka Holding AG, Buchs, Switzerland NMPN-methylpyrrolidinone TEA Triethylamine TPEG 990 A glycerin-startedtrifunctional polyethylene glycol M_(n) approximately 990 commerciallyavailable from Dow Chemical Company, Midland, MI THF Tetrahydrofuran Nasaccharin Sodium salt of saccharin, dehydrated

Preparative Example 1

In a glass reaction vessel, a mixture of DMF (154 milliliters),4-carboxybenzenesulfonamide (30.0 grams), succinic anhydride (16.41grams), and triethylamine (33.19 grams) was stirred and heated to 50° C.under a nitrogen atmosphere for four hours. The mixture was allowed tocool to room temperature, acetic anhydride (18.27 milliliters) was addedand the mixture was stirred at room temperature for an additional threehours. The mixture was poured into 400 milliliters of stirred 1N aqueousHCl. This mixture was filtered, washed with deionized water and dried ina vacuum oven to afford the desired product. Yield: 36.94 grams.

Preparative Example 2

In a glass reaction vessel containing a stirred mixture of thecarboxy-containing product of Preparative Example 1 (20.0 grams) and dryacetonitrile (85 grams) was added thionyl chloride (10.0 grams) and DMF(1 drop). The resulting mixture was stirred and heated under reflux forone hour, cooled to room temperature and further cooled in an ice bath,which resulted in the formation of a solid precipitate. The solid wascollected by filtration, washed sequentially with cold acetonitrile andcold toluene, and dried overnight in a vacuum oven at 50° C. to give thedesired product. Yield: 17.7 grams.

Preparative Example 3

In a glass reaction vessel, a mixture of norbornene-2,3-dicarboxylicanhydride (26.9 grams), 4-carboxybenzenesulfonamide (30.0 grams), TEA(49.8 grams) and DMF (82 grams) were stirred and heated to 50° C. undera nitrogen atmosphere for two hours followed by heating overnight at 90°C. The mixture was cooled to room temperature and acetic anhydride (18.3grams) was added to the flask. The mixture was stirred overnight at roomtemperature, poured into aqueous 1N HCl and the resultant solid wasisolated by filtration and dried using a vacuum oven. The resultingsolid was recrystallized from glacial acetic acid to give the desiredproduct. Yield: 12.6 grams.

Preparative Example 4

In a glass reaction vessel fitted with a reflux condenser and a nitrogeninlet, a mixture of the carboxylic acid product of Preparative Example 3(5.0 grams), thionyl chloride (2.2 grams), DMF (1 drop) and ACN (28.9milliliters) were stirred under a nitrogen atmosphere and heated toreflux for one hour. The mixture was allowed to cool to room temperatureand the volatile components were removed using a rotary evaporator. Theresultant solid was washed into a fritted glass funnel, washed withdiethyl ether, and then dried at room temperature under a stream ofnitrogen gas to afford the desired product. Yield: 4.7 grams.

Example 1 Preparation of

Sebacoyl chloride (6.0 grams, 0.025 mol) was added to a stirred slurryof dry Na saccharin (10.25 grams, 0.50 mol) and 200 milliliters ofacetone under a nitrogen atmosphere. The resulting mixture was stirredfor 20 hours at room temperature. IR spectroscopy showed the absence ofpeaks for the group —C(O)Cl. The mixture was filtered and washed withacetone to give 11.8 grams of white solid. The acetone was removed fromthe wash solution to yield 3.7 grams of a tan solid that was combinedwith the filtrate, washed with water and dried to give the desiredproduct (structure confirmed by NMR) which was slightly soluble in ACN,acetone and 2-butanone. Yield: 9.1 grams.

Example 2 Preparation of

Ten milliliters of SOCl₂ was added to a mixture of PEG 600 diacid (30grams, 0.05 mol, where m′ is approximately 14) in 100 milliliters CH₂Cl₂with immediate evolution of HCl. After 20 hours, the solvent was removedunder vacuum to give 33.6 grams of pale yellow oil. Of this, 6.4 grams(0.01 mol) was added to dry Na saccharin (4.1 grams, 0.02 mol). Theresulting slurry was stirred for 24 hours, filtered and dried undervacuum to give the desired product as a pale tan syrup. Yield: 9.3grams.

Example 3 Preparation of

In a glass reaction vessel, a sample of the chlorocarbonyl product ofPreparative Example 2 (1.0 grams) was dissolved in NMP (3.6 grams) andchilled in an ice bath. A solution of PEG 3400 (3.54 grams, where m′ isapproximately 77) in THF (3.54 grams) was slowly added to the flask. Themixture was stirred overnight as the mixture warmed to room temperature.The mixture was concentrated, recrystallized with IPA. The resultingwhite solid was filtered and rinsed with chilled IPA to give the desiredproduct. Yield: 4.17 grams. A hydrogel was formed by the addition ofpolyethylenimine, average M_(w) approximately 2,000, 50 wt. % solutionin water (0.17 grams) to an aqueous solution of this product (0.50grams), at 50 weight percent.

Example 4 Preparation of

In a glass reaction vessel, a sample of the chlorocarbonyl product ofPreparative Example 4 (1.99 grams) was dissolved in THF (10 grams). Asolution of PEG 3400 (8.20 grams, where m′ is approximately 77),pyridine (0.48 grams), and THF (3.54 grams) were slowly added and theresulting mixture was stirred overnight. The mixture was concentratedand recrystallized with IPA. The resulting white solid was filtered andrinsed with chilled IPA to give the desired product. Yield: 9.0 grams.

Example 5 Preparation of

In a glass reaction vessel, a sample of the chlorocarbonyl product ofPreparative Example 2 (2.00 grams) was dissolved in THF (8 grams). Asolution of TPEG 990 (2.07 grams, where m′ is approximately 22), TEA(0.70 grams), and THF (8.0 grams) were slowly added and the resultingmixture was stirred overnight. IR spectroscopy showed the absence ofpeaks for the group —C(O)Cl. The mixture was concentrated, reconstitutedin EtOAc, and filtered to give a clear, colorless solution at 24.5%solids.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

1. A compound of the formula:(A-)_(y)-Q wherein each A is independently selected from the groupconsisting of functional groups having the following formulas:

wherein: R¹ and R² together with a dicarboximide group to which they areattached form a four to eight membered heterocyclic or heterobicyclicgroup that can be fused to an optional aromatic group, optionalsaturated or unsaturated cyclic group, or optional saturated orunsaturated bicyclic group; R³ is an alkyl, aryl, aralkyl, or—NR^(a)R^(b) wherein R^(a) and R^(b) are each an alkyl group or takentogether with the nitrogen atom to which they are attached form a fourto eight membered heterocyclic group; R⁴ and R⁵ together with adicarboximide group to which they are attached form a four to eightmembered heterocyclic or heterobicyclic group that can be fused to anoptional aromatic group, optional saturated or unsaturated cyclic group,or optional saturated or unsaturated bicyclic group; Y¹ and Y² are eachindependently a single bond or a divalent group selected from the groupconsisting of an alkylene, heteroalkylene, arylene, heteroarylene,carbonyl, carbonyloxy, carbonylimino, oxy, thio, —NR^(f)— where R^(f) ishydrogen or alkyl, and combinations thereof; Q is a single bond or any-valent atom or group; and y is an integer of 2 to 10; with the provisothat Q, Y¹, and Y² are free of disulfide groups.
 2. The compound ofclaim 1 wherein Y¹ and Y² are each independently selected from the groupconsisting of groups having the following formulas: —Y^(1a)—Ar¹— and—Ar¹—Y^(1a)—, wherein: Ar¹ is an arylene; and Y^(1a) is selected fromthe group consisting of a single bond, alkylene, heteroalkylene,carbonyl, carbonyloxy, carbonylimino, oxy, thio, —NR^(f)— where R^(f) ishydrogen or alkyl, and combinations thereof.
 3. The compound of claim 1wherein Y¹ and Y² each independently comprises a first alkylene grouplinked to an arylene group with a group selected from the groupconsisting of a carbonyl, carbonyloxy, carbonylimino, oxy, thio,—NR^(f)— where R^(f) is hydrogen or alkyl, and combinations thereof. 4.The compound of claim 1 wherein Y¹ and Y² each independently comprises afirst heteroalkylene group linked to an arylene with a group selectedfrom the group consisting of a carbonyl, carbonyloxy, carbonylimino,oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, and combinationsthereof.
 5. The compound of claim 1 wherein Y¹ and Y² each independentlycomprises a first alkylene group connected to a second alkylene group orto a first heteroalkylene group with a group selected from the groupconsisting of a carbonyl, carbonylimino, carbonyloxy, oxy, thio,—NR^(f)— where R^(f) is hydrogen or alkyl, and combinations thereof. 6.The compound of claim 1 wherein Y¹ and Y² are each independently aheteroalkylene group.
 7. The compound of claim 1 wherein Y¹ and Y² eachindependently comprises a first heteroalkylene group connected to asecond heteroalkylene group or to a first alkylene group with a groupselected from the group consisting of a carbonyl, carbonylimino,carbonyloxy, oxy, thio, —NR^(f)— where R^(f) is hydrogen or alkyl, andcombinations thereof.
 8. The compound of claim 1 wherein Y¹ and Y² areeach independently a heteroalkylene having 1-30 carbon atoms and up to30 heteroatoms selected from the group consisting of N, O, S, andcombinations thereof, wherein the heteroalkylene group is linear,branched, cyclic, or combinations thereof.
 9. The compound of claim 1wherein Y¹ and Y² are each independently an alkylene having 1-30 carbonatoms, wherein the alkylene group is linear, branched, cyclic, orcombinations thereof.
 10. The compound of claim 1 wherein Y¹ and Y² eachindependently comprises an arylene group, in addition to one or morealkylene groups and one or more heteroalkylene groups.
 11. The compoundof claim 1 wherein each A is independently selected from the groupconsisting of functional groups having the following formulas:

wherein each R is independently an alkyl group.
 12. The compound ofclaim 1 wherein each A is independently selected from the groupconsisting of functional groups having the following formulas:

wherein: each n is independently an integer of 1 to 100; m is an integerof 1 to 200; k is an integer of 2 to 4; q in an integer of 0 or 1; D isoxygen, sulfur, or NH; Ar¹ is an arylene group; and each L isindependently oxygen or NR^(f) where R^(f) is hydrogen or alkyl.
 13. Thecompound of claim 1 wherein each A is independently selected from thegroup consisting of functional groups having the following formulas:

wherein: v is an integer of 1 to 200; x is an integer of 1 to 4; and Dis oxygen, sulfur, or NH.
 14. The compound of claim 1 wherein R³ is analkyl, aryl, or aralkyl.
 15. The compound of claim 1 wherein Q is anatom selected from C, N, S, O, or P, or is a y-valent group containingup to 20 carbon atoms and up to 6 heteroatoms and/or functional groups.16. The compound of claim 1 wherein y is 2 or 3.